1. Field of the Invention
The present invention generally relates to inhibition of proliferation and function of lymphocytes and other cells of lymphoid origin which contain the Janus tyrosine kinase (Jak3). More particularly the invention relates to therapeutic and testing methods using chemical agents which block lymphocyte function, especially regulation of immune activity. Still more particularly, the invention relates to selectively disrupting Janus tyrosine kinase 3 (Jak3) mediated cell activities and cell proliferation.
2. Description of Related Art
The efficacy of therapeutic strategies in use today to combat organ allograft rejection is severely limited due to dependence on immunosuppressive drugs that produce potent side effects. Current clinical immunosuppressive regimens are dominated by the serine-threonine phosphatase calcineurin (CaN) inhibitors cyclosporin A (CsA) and tacrolimus (FK506),1 which act as T-cell modifiers by blocking T-cell progression through the early G1 stages of the cell cycle.1,2 Undesirable side effects associated with those drugs include nephrotoxicity, neurotoxicity, diabetes mellitus, hyperlipidemia, hypertension, hirsutism, and gingival hyperplasia.3 A newer drug, rapamycin (RAPA), which targets the serine-threonine kinase mammalian target of RAPA (mTOR), can also manifest mucosal ulcers, lymphoproliferative disorders, hypokalemia, and increases in low density lipoproteins, cholesterol, and triglycerides.4 A serious drawback of the clinically approved drugs is that they do not yield permanent acceptance of allografts, and therefore they need to be continuously delivered to the patients.
Recent therapeutic strategies to combat organ allograft rejection have focused on T-cell signaling pathways and the molecules that comprise them. T-cell signaling cascades and their potential role in immunosuppression and potentially for the induction of transplantation tolerance are described by Kirken and Stepkowski.5 Complete activation of T-cells requires three threshold-limited sequential signals.6 
Signal 1 delivered by antigens that engage a specific T-cell receptor (TCR) is then followed by signal 2 delivered by a B7/CD28 interaction. Within seconds to minutes after TCR engagement, the CD3ζ chain is tyrosine (Tyr) phosphorylated during the autoactivation of Zap70, Lck, and Fyn protein Tyr kinases.7-9 Concomitantly, calcium (Ca2+) mobilization triggers catalytic activation of CaN phosphatase to dephosphorylate nuclear factor of activated T-cell (NFAT)—a necessary step for NFAT to translocate to the nucleus and bind discrete DNA binding elements within the promoter of the interleukin (IL)-2 gene.10 Signals 1 and 2 are critical for the synthesis and secretion of IL-2, which, in concert with other T-cell growth factors (TCGFs) such as IL-4, -7, -9, -13, -15 and -21, deliver signal 3 through cytokine receptors, a necessary step required to drive clonal expansion of T-cells.11 These cytokine receptors share a common γ chain (γc) that when combined with a unique α-chain for each cytokine deliver intracellular signals via Janus tyrosine (Tyr), Jak1 and Jak3, as well as activate signal transducers and activators of transcription (Stat)1, Stat3, Stat5a/b and Stat6.11-18 
The CaN enzyme (participating in the signal 1 pathway in T-cells) and the mTOR enzyme (participating in the signal 3 pathway in T-cells) are ubiquitously expressed in various tissues throughout the body. This severely limits the efficacy of their inhibitory drugs such as CsA, FK506 and RAPA for T-cell specific targeting. RAPA is the only effective signal 3 inhibitor that has been clinically approved to date24. Unlike other signaling pathway molecules that serve as candidate targets for therapeutic intervention, Jak3 expression shows a limited pattern of tissue expression and is compartmentalized to T-cells, B-cells, natural killer (NK) cells and monocytes, or in general terms to cells of immune origin.
Due to its primary localization to lymphoid-type cells, Jak3 holds promise as a unique molecular and therapeutic target for ablating a number of immune-derived diseases.19-21 This enzyme is almost exclusively associated and activated via γc, and therefore genetic disruption of Jak3 or γc is manifested as severe combined immunodeficiency disease.22 The reasons for this profound immune suppression is due to Jak3's critical role in T-cell development and recruitment by a family of TCGFs as mentioned above. Because Jak3 is associated with the receptor component and membrane proximal, all downstream signals emanating from these receptors, including Stat and mitogen activated protein kinase (Mapk) cascades would be activated. Thus, disruption of Jak3 subsequently blocks all signals mediated by TCGF and hence their ability to regulate gene transcription within these cells. However, if one could control the inhibition of this unique and redundant signaling pathway, favorable regulation of immune activity should be attainable as observed in patients and mice defective in these genes. Moreover, targeting this pathway would, in theory, also inhibit a population of activated and proliferating T-cells responsible for rejection that are not responsive to CsA21 
Efforts to identify inhibitors that specifically target Jak3 in lymphocytes are hampered by the fact that the few reported drugs that inhibit Jak3 also inhibit a plethora of other tyrosine kinases that are required for routine cell function in many body tissues. Indeed these protein tyrosine kinases are fundamentally important for transducing extracellular signals from cell surface receptors to the nucleus, subsequently regulating growth, differentiation and function in cell types other than lymphocytes.
U.S. Patent Application Publication 2002/0042513 (Uckun et al.) describes certain quinazoline compounds that are selected on the basis of their estimated docking affinity using a Jak3 homology model based on structural homology to the insulin receptor tyrosine kinase. The ability of some quinazoline compounds to treat or prevent transplant complications, autoimmune induced diabetes, or to prolong allograft survival were evaluated.
U.S. Patent Application Publication No. 2002/0032204 (Moon et al.) describes certain Mannich base prodrugs of certain 3-(pyrrol-2-ylmethylidene)-2-indolinone derivatives that modulate the catalytic activity of receptor tyrosine kinases (RTKs), non-receptor protein tyrosine kinases (CTKs) and serine/threonine protein kinases (STKs). These prodrugs are said to be useful for treating many diseases mediated by abnormal protein kinase activity. The disclosed compounds are said to modulate RTK, CTK and/or STK mediated signal transduction pathways as a therapeutic approach to cure many kinds of solid tumors. Other Mannich base compounds have been described and evaluated for cytotoxicity and anticancer properties.34,35 Certain Mannich bases of conjugated styryl ketones with antifungal and antineoplastic properties are the subject of U.S. Pat. No. 6,017,933.
Recently, two agents that show selectivity for Jak3 have been identified.21,24,25 One agent denoted as AG-490 is a tyrphostin family member and a derivative of benzylidene malononitrile, which has the structural formula:

Another is PNU156804, which is a congener of the toxic parent compound undecylprodigiosin, and has the structural formula:

Both AG-490 and PNU156804 are competent to inhibit T-cell proliferation mediated by TCGFs and Jak3 autokinase activity, while having limited effects on unactivated T-cells that fail to express Jak3. It was found that neither of those two agents affects T-cell receptor activation cascade intermediates including p56Lck or Zap70 tyrosine kinases.23,25 
In one study AG-490 treatment reduced graft infiltration of mononuclear cells (GICs) and Stat5a/b DNA binding of ex vivo IL-2 stimulated GICs, but failed to affect IL2Rα expression as judged by ribonuclease protection assays. Thus, it was concluded that inhibition of Jak3 prolongs allograft survival and also potentiates the immunosuppressive effects of CsA, but not RAPA. It was also found that AG-490 does not inhibit other tyrosine kinase family members Lck, Lyn, Btk, Syk, Src, Jak1 or Tyk2 kinases, while it does exert similar effects on its closest related family member Jak2.24 Adverse side effects of AG-490 preclude routine clinical use for immunosuppressive therapy.
PNU156804, on the other hand, displayed greater specificity by inhibiting Jak3 mediated T-cell growth by IL-2 as compared to other growth factors (prolactin) that use Jak2. Kinase assays showed that PNU156804 preferentially inhibited Jak3 autophosphorylation, as compared with Jak2, as well as shared intermediate effector molecules such as the Stat5 pathways.25 In that study, it was shown that PNU156804 prolongs allograft survival and acts synergistically with CsA but additively with RAPA. It was also established that PNU156804 preferentially disrupts Jak3 (compared with Jak2 autokinase activity), thereby selectively inhibiting γc-driven T-cell clonal expansion. Current models hold that Jak3 is an upstream activator of mTOR. Since Jak3 is expressed in immune cells, inhibition of Jak3 will block mTOR activation without the adverse effects currently associated with RAPA. Moreover, synergy between CsA (blocking G0-G1 transition) and PNU156804 (blocking G1-S progression) offers a novel strategy for immunosuppression by blocking sequential activation signals thereby requiring lower dosages of each drug while maintaining a beneficial therapeutic effect.25 PNU156804 has subsequently proven to be too toxic for therapeutic use in humans, however.
While some currently available drugs have shown promise in blocking acute rejection, the problems of chronic graft destruction and permanent allograft acceptance (e.g. transplantation tolerance) in the absence of continuous immune suppression remain unabated. Therapeutic methods that adequately address these problems and pharmaceuticals that avoid adverse side effects are needed. Toward those goals, great strides have been made in understanding T-cell signal transduction and in devising strategies for targeting certain molecules in the signaling pathways. There is an urgent need for agents that selectively or specifically inhibit molecules unique for signal 3 pathways of T- and B-lymphocytes activated by TCGFs. Such agents have great potential for blocking clonal expansion of T-cells without affecting other cells. As discussed above, Jak3 represents a unique molecular target in the signal 3 pathway for regulating unwanted immune responses such as host-versus-graft and graft-versus-host disease.